1. Field of the Invention
This invention relates to opioid receptors from mammalian species and ligands specific for such receptors. Specifically, the invention relates to the isolation of an endogenous peptide ligand specific for a novel mammalian opioid receptor. The invention also relates to the construction of analogues, derivatives and peptide mimetics of this endogenous mammalian opioid receptor ligand. Specifically provided is a mammalian hypothalamus-derived endogenous opioid receptor ligand, synthetic embodiments and analogues thereof. Methods of making and using such ligands, as well as antibodies against and epitopes of this novel opioid receptor ligand are also provided by the invention.
2. Background of the Invention
The use (and abuse) of opiates, archetypally opium and morphine, have been known since antiquity (reviewed in Brownstein, 1993, Proc. Natl. Acad. Sci. USA 90: 5391-5393). Since the nineteenth century, chemical characterization and synthesis of a number of morphine analogues have been achieved in an effort to discover a compound with the analgesic effects of morphine that lacks or is substantially attenuated in its addictive potential. These efforts have proven fruitless to date.
The biology behind the reasons why morphine and morphine-like compounds display both analgesic and addictive properties was first elucidated by the discovery of endogenous morphine-like compounds termed enkephalins (see DiChara and North, 1992, Trends in Pharmacol. Sci. 13: 185-193 for review). Accompanying this finding of an endogenous opiate was the biochemical evidence for a family of related but distinct opiate receptors, each of which displays a unique pharmacological profile of response to opiate agonists and antagonists (see McKnight and Rees, 1991, Neurotransmissions 7: 1-6 for review). To date, four distinct opiate receptors have been described by their pharmacological profiles and anatomical distribution: these comprise the xcexc, xcex4, xcexa and "sgr" receptors (the "sgr" receptor has been determined to be a non-opioid receptor with cross-reactivity to some opioid agonists).
Thus, mammalian opioid receptors are known in the art, and some of these proteins have been isolated biochemically and their corresponding genes have been recently cloned using genetic engineering means.
Kieffer et al., 1992, Proc. Natl. Acad. Sci. USA 89: 12048-12052 disclosed the isolation of a cDNA copy of the mouse xcex4-opioid receptor by expression cloning.
Evans et al., 1992, Science 258: 1952-1955 disclose the isolation of a cDNA copy of the mouse xcex4-opioid receptor by expression cloning.
Chen et al., 1993, Molec. Pharmacol. 44: 8-12 disclose the isolation of a cDNA copy of the rat xcexc-opioid receptor.
Yasuda et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6736-6740 disclose the isolation of a cDNA copy of each of the mouse K- and xcex4-opioid receptor.
Bzdega et al., 1993, Proc. Natl. Acad. Scd. USA 90: 9305-9309 disclose the isolation and chromosomal location of the xcex4-opioid receptor in the mouse.
The present inventors have cloned, expressed and functionally characterized a novel mammalian opioid receptor gene, disclosed in co-owned and co-pending U.S. patent application, Ser. No. 08/149,093, filed Nov. 8, 1993, and issued as U.S. Pat. No. 5,658,783 on Aug. 19, 1997 which is hereby incorporated by reference in its entirety. Specifically disclosed therein are nucleic acids encoding the novel mammalian opioid receptor gene, recombinant expression constructs comprising this opioid receptor gene, cells containing such constructs and expressing the novel opioid receptor gene, and methods for making and using such nucleic acids, constructs and cells for opioid detection and novel drug screening. The nucleic acid sequence of the MSOR gene and the deduced amino acid sequence of the cognate receptor protein were also disclosed in this prior application.
In 1991, U.S. pharmaceutical companies spent an estimated $7.9 billion on research and development devoted to identifying new therapeutic agents (Pharmaceutical Manufacturer""s Association). The magnitude of this amount is due, in part, to the fact that the hundreds, if not thousands, of chemical compounds must be tested in order to identify a single effective therapeutic agent that does not engender unacceptable levels of undesirable or deleterious side effects. There is an increasing need for economical methods of testing large numbers of chemical compounds to quickly identify those compounds that are likely to be effective in treating disease.
This is of particular importance for psychoactive and psychotropic drugs, due to their pharmacological importance and their potential to greatly benefit or greatly harm human patients treated with such drugs. At present, few such economical systems exist. Conventional screening methods require the use of animal brain slices in binding assays as a first step. This is suboptimal for a number of reasons, including interference in the binding assay by non-specific binding of heterologous (i.e., non-receptor) cell surface proteins expressed by brain cells in such slices; differential binding by cells other than neuronal cells present in the brain slice, such as glial cells or blood cells; and the possibility that putative drug binding behavior in animal brain cells will differ from the binding behavior in human brain cells in subtle but critical ways. For these and other reasons, development of in vitro screening methods for psychotropic drugs has numerous advantages and is a major research goal in the pharmaceutical industry. The ability to synthesize human opioid receptor molecules in vitro would represent one way to provide an efficient and economical means for rational drug design and rapid screening of potentially useful compounds.
A great advantage in efforts for developing novel psychotropic drugs which exert their activity (analgesic and otherwise) aria binding to mammalian opioid receptors would be to identify the endogenous ligand(s) which bind to such receptors. Certain such ligands have been isolated in the prior art, including the peptides comprising the endorphins and enkephalins (see Jaffe and Martin, 1990, xe2x80x9cOpioid Analgesics and Antagonistsxe2x80x9d, in Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, 8th ed. (Pergammon Press, Inc.: New York), Chapter 21, p.485-521). The identification and characterization of additional endogenous ligands would advantageously provide another basis for rational drug design and an appreciation for structural features of such ligands both shared with other opioid receptor ligands and unique for ligands specific for individual receptors or subclasses of receptors.
This invention provides small, readily-produced peptides that are ligands for a novel mammalian opioid receptor protein having the amino acid sequence identified as SEQ ID Nos.: 5 and 6. Peptides of the invention are characterized as having an amino acid sequence that is the amino acid sequence identified as SEQ ID Nos.: 5 and 6 or a subsequence thereof, amino acid sequence variants of the sequence or subsequence, as well as analogues and derivatives thereof, that are ligands for the novel mammalian opioid receptor protein having the amino acid sequence identified as SEQ ID Nos.: 5 and 6, as well as analogues and derivative thereof.
The peptides of the invention include linear and cyclized peptides, and synthetic analogues and variants thereof. Certain embodiments of such variants include, substitution variants, wherein an amino acid residue at one or more positions in the peptide is replaced with a different amino acid residue (including atypical amino acid residues) from that found in the corresponding position of amino acid sequence of the parent peptide of the invention. In a preferred embodiment, the substituted amino acid is tyrosine. Certain other embodiments of peptide variants of the invention include addition variants, wherein such variant peptides may include up to about a total of 10 additional amino acids, covalently linked to either the amino-terminal or carboxyl-terminal extent, or both, of the parent, opioid receptor binding peptide of provided by the invention. Such additional amino acids may also include atypical amino acids. Linear and cyclized embodiments of the amino acid substitution and addition variant peptides are also provided as peptides of the invention. In addition, peptides of the invention may be provided as fusion proteins with other functional targeting agents, such as immunoglobulin fragments. Derivatives of the peptides of the invention also include modifications of the amino- and carboxyl-termini and amino acid side chain chemical groups such as amines, carboxylic acids, alkyl and phenyl groups.
In a first aspect, the invention provides peptides of the formula: (Xaa)n-Phe-Gly-Gly-Phe-(A1)-(A2)-(A3)-(A4)-(A5)-(A6)-(A7)-(A8)-(A9)-(A10)-(A11)-(A12)-Gln-(Xaa)m wherein A1 is Thr, Leu or Met; A2 is Gly, Arg or Thr; A3 is Ala, Arg or Ser; A4 is Arg, Ile, Glu or Gln; A5 is Lys, Arg or Phe; A6 is Ser, Pro or Lys; A7 is Ala, Lys, Gln or Val; A8 is Arg, Leu, Thr or Val; A9 is Lys, Pro or Thr; A10 is Tyr, Leu or Trp; A11 is Ala, Asp or Val; A12 is Asn. or Thr; (Xaa) is any amino acid; n and m are integers wherein n+m is no more than 82(SEQ ID No.:15); and the amino acids are each individually in either the D or L stereochemical configuration and the peptide specifically binds to a mammalian opioid receptor having an amino acid sequence identified by Seq ID Nos.:4. In a preferred embodiment, the peptide has the formula:
Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Glnxe2x80x83xe2x80x83(SEQ ID No.:5),
and n+m equals zero. In an another preferred embodiment, the peptide has the formula:
Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Tyr-Ala-Asn-Glnxe2x80x83xe2x80x83(SEQ ID No.:6),
and n+m equals zero. Both naturally-occurring embodiments of such peptides, purified using well-established techniques from the cells or tissues producing the peptide, and synthetic embodiments, are within the scope of the invention.
In other embodiments of this aspect of the invention are provided peptides of general formula:
(Xaa)n-Phe-Gly-Gly-Phe-(A1)-(A2)-(A3)-(A4)-(A5)-(A (A6)-(A7)-(A8)-(A9)-(A10)-(A11)-(A12)-Gln-(Xaa)m
and having amino acid substituents as described above wherein such peptides are radiolabeled by conjugation with or binding to a radioactive isotope. In a preferred embodiment, the peptide has the formula:
Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Tyr-Ala-Asn-Glnxe2x80x83xe2x80x83(SEQ ID No.:6),
n+m equals zero, and the radiolabel is a radioisotope of iodine. In other preferred embodiments, the peptide is covalently linked to a radiolabel binding moiety and the radiolabel a radioisotope of indium, gallium, technetium, rhenium or other useful radioisotope.
The invention also provides pharmaceutical compositions of the peptides of the invention. In a first aspect, the peptides of the invention are provided in a pharmaceutical composition comprising an acceptable carrier or diluent. In a second aspect, such pharmaceutical compositions are provided in detectably-labeled embodiments, useful for diagnostic identification of sites of both normal and pathological peptide ligand receptor binding in vivo and in vitro. In such embodiments, the peptides of the invention are detectably labeled, for example, with a radioisotope such as I-123, 1-125 or I-131, conjugated to the peptide via, inter alia, a tyrosine residue that is non-essential for receptor binding. Additional radioisotopes, such as In-111, Ga-67, Re-186, Re-188 and Tc-99m, can be conjugated to such peptides using methods well-understood in the art. In such embodiments, the pharmaceutical composition is radiolabeled to an appropriate specific activity, and administered to an animal, preferably a human, at a diagnostically-effective and non-toxic dose. Methods and routes of administration may be selected by those with skill in the art based on well-established techniques and in a clinically-appropriate fashion.
In a second aspect of the pharmaceutical compositions of the invention, the opioid receptor-binding ligand peptides are provided in an appropriate composition with acceptable carriers of diluents and in a therapeutically-effective amount. In such embodiments, the therapeutic pharmaceutical compositions are used in methods to treat diseases or pathological conditions associated with ligand binding to the MSOR receptor. Specifically provided are methods for treating locomotor diseases in an animal.
The invention also provides methods for designing MSOR opioid receptor ligands and analogues, derivatives and mimetics thereof, using the amino acid sequence of the peptide ligands of the invention. Also encompassed within the scope of this invention are such analogues, derivatives and mimetics produced by the methods of the invention.
In a second aspect, the invention provides nucleic acids encoding a novel mammalian opioid-specific receptor protein having an amino acid sequence identified as SEQ ID No.:4, recombinant eukaryotic expression constructs capable of expressing the novel mammalian opioid-specific receptor of the invention in cultures of transformed cells, as well as cultures of transformed eukaryotic cells that synthesize the receptor of the invention. The invention also provides homogeneous compositions of the receptor protein having an amino acid sequence identified as SEQ ID No.:4, and antibodies against and epitopes of the receptor protein of the invention. Methods for characterizing these receptor proteins and methods for using these proteins in the development of agents having pharmacological uses related to these receptors are also provided by the invention.
In this aspect of the invention is provided a nucleic acid having a nucleotide sequence encoding a mammalian MSOR opioid receptor. In a preferred embodiment, the nucleic acid encodes 1452 nucleotides of the cDNA comprising 1101 nucleotides of coding sequence, 181 nucleotides, of 5xe2x80x2 untranslated sequence and 170 nucleotides of 3xe2x80x2 untranslated sequence, depicted in FIGS. 1A and 1B and identified as SEQ ID No:3. Encompassed in this aspect of the invention is the disclosed sequence and allelic variants of this sequence, either naturally occurring or the product of in vitro chemical or genetic modification.
The corresponding receptor protein, having the deduced amino acid sequence shown in FIGS. 1A and 1B and identified as SEQ ID No.:4, is also an aspect of the invention. In particular embodiments of this aspect of the invention is comprised a homogeneous composition of the 47 kD mammalian opioid receptor protein and derivatives thereof, said size being understood to be the size of the protein before any post-translational modifications. The amino acid sequence of this 47 kD receptor protein is depicted in FIGS. 1A and 1B and identified as SEQ ID No:4.
This invention provides both nucleotide and amino acid sequence probes derived from the sequences herein provided, isolated from either cDNA or genomic DNA, as well as probes made synthetically with the sequence information derived therefrom. The invention specifically includes but is not limited to oligonucleotide, nick-translated, random primed, or in vitro amplified probes made using cDNA or genomic clones embodying this aspect of the invention, and oligonucleotide and other synthetic probes synthesized chemically using the nucleotide sequence information of cDNA or genomic clone embodiments of the invention.
The present invention also includes synthetic peptides made using the nucleotide sequence information comprising the cDNA embodiments of the receptor protein embodiments of the invention. The invention includes either naturally occurring or synthetic peptides which may be used as antigens for the production of receptor-specific antibodies, or useful as competitors of receptor molecules for agonist, antagonist or drug binding, or to be used for the production of inhibitors of the binding of agonists or antagonists or analogues -thereof to such MSOR receptor molecules. Particularly preferred embodiments of this aspect of the invention are such peptides that interact with the peptide ligand embodiments of the invention and enhance or inhibit peptide ligand binding to the receptor protein.
The present invention also provides antibodies against and epitopes of the mammalian opioid receptor molecules of the invention, including antisera and both polyclonal and monoclonal embodiments of such antibodies and hybridoma cell lines producing such monoclonal antibodies.
The present invention provides recombinant expression constructs comprising a nucleic acid encoding a mammalian MSOR receptor of the invention wherein the construct is capable of expressing the encoded MSOR receptor in cultures of cells transformed with the construct. Preferred embodiments of such constructs comprise the MSOR receptor cDNA depicted in FIGS. 1A and 1B (SEQ ID No.:3), such constructs being capable of expressing the MSOR receptor encoded therein in cells transformed with the construct.
The invention also provides cultures cells transformed with the recombinant expression constructs of the invention, each such cultures being capable of and in fact expressing the mammalian MSOR receptor encoded in the transforming construct.
The present invention also includes within its scope protein preparations of prokaryotic and eukaryotic cell membranes containing the MSOR receptor protein of the invention, derived from cultures of prokaryotic or eukaryotic cells, respectively, transformed with the recombinant expression constructs of the invention.
The invention also provides methods for screening compounds for their ability to inhibit, facilitate or modulate the biochemical activity of the mammalian MSOR receptor molecules of the invention, for use in the in vitro screening of novel agonist and antagonist compounds. In preferred embodiments, cells transformed with a recombinant expression construct of the invention are contacted with such a compound, and the binding capacity of the compounds, as well as the effect of the compound on binding of other, known opioid agonists and antagonists, is assayed. Additional preferred embodiments comprise quantitative analyses of such effects. The invention specifically provides a method for screening a compound for a capacity to bind to a mammalian MSOR opioid receptor in cells expressing the receptor, using a competitive assay between the compound to be tested and the peptide ligands of the invention. The method comprises the steps of:
(a) transforming a culture of eukaryotic or prokaryotic cells with a recombinant expression construct capable of expressing a mammalian MSOR opioid receptor having an amino acid sequence identified as SEQ ID No.:4, wherein the cells of the transformed cell culture express the opioid receptor;
(b) assaying the transformed cell culture for binding of an amount of a detectably-labeled peptide according to claim 1 in competition with varying amounts of the compound; and
(c) determining whether the compound competitively binds to the opioid receptor by calculating the extent of inhibition of binding of the detectably-labeled peptide in the presence of the compound.
In additional embodiments of this method is included the additional step of:
(d) comparing the binding capacity of the compound with the binding capacities of additional compounds that are known to bind to mammalian opioid receptors, wherein said additional compounds comprise naturally-occurring and synthetic opioid receptor agonists and antagonists.
The present invention is also useful for the detection of analogues, agonists or antagonists, known or unknown, of the mammalian MSOR receptors of the invention, either naturally occurring or embodied as a drug. In preferred embodiments, such analogues, agonists or antagonists may be detected in blood, saliva, semen, cerebrospinal fluid, plasma, lymph, or any other bodily fluid. In particularly preferred embodiments, the peptide ligands of the invention are used in competitive binding assays to quantitatively evaluate novel ligand binding to the MSOR receptor.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.